Osteopontin (OPN) is a secreted phosphoprotein that is associated with cancer, cardiovascular disease, renal injury and inflammation. One of the first descriptions of osteopontin was as a secreted phosphoprotein (spp 1) found at elevated levels circulating in the serum of cancer patients. Since that time, numerous studies have shown upregulated expression of osteopontin in various human cancers. Not limited to cancer, osteopontin expression is associated with various tissue injury and human diseases. Several clinical studies have been performed to assess the potential of osteopontin to serve as a clinical marker for disease progression or prognosis in human breast cancer. The results of these studies show that there is a significant overlap of osteopontin with other variables associated with patient outcome including high histological grade, c-ErbB3 and p53 (Rudland, et al., Cancer Res, 62(12):3417-3427, 2002). Significantly, these breast cancer patients were studied for 14-20 years of follows up and, while the low-osteopontin group had a median survival of >228 months, the high-osteopontin group had a median survival of 68 months, suggesting predictive value of osteopontin levels in long-term patient outcome. Other studies suggest that, in addition to breast cancer, elevated osteopontin in the serum is associated with prostate and lung cancer (Fedarko, et al., Clin Cancer Res 7(12):4060-4066, 2001).
There are several characteristics of osteopontin that make it a valuable candidate as a biomarker for disease. The protein is secreted in body fluids and can be found in, e.g., plasma and serum, human milk and urine. There are available ELISA-based assays to test for osteopontin protein levels. However, multiple modified fragments of osteopontin exist that represent proteolytically cleaved fragments of the parent molecule. These cleaved fragments are distinct functionally (Gao, Y. A., et al., Matix Biol, 23(7):457-466, 2004; Senger, D. R., et al., Ann NY Acd Sci, 760:83-100, 1995; Senger, D. R., et al., Am J Pathol, 149(1):293-305, 1996; Senger D. R., et al., Biochem Biophys Acta, 1314(1-2):13-24, 1996; Bayless, K. J., et al., J Biol Chem, 276(16):13483-13489, 2001; Green, P. M., et al., FEBS Lett, 503(1):75-79, 2001; Smith, L. L., et al., Exp Cell Res, 242:351-360, 1998; Yokosaki, Y., et al., Matrix Biol, 24(6):418-427, 2005), they occur in vivo and are generated through the catalytic activity of proteases such as thrombin and the matrix metalloproteinases that are known to be associated with tumor progression. Indeed, the question of whether these osteopontin fragments wound provide a more accurate assessment of clinical tumor burden, progression or patient outcome is an important issue that has not been addressed. Part of the limitation is that there are not reagents that will specifically detect osteopontin fragments and distinguish them from each other and the full-length protein. Thus far only SDS-PAGE analysis followed by immunoblotting with anti-osteopontin antibodies can show fragment presence in clinical samples. However, even this method has significant drawbacks since antibodies for specific OPN fragments may not exist. For example, a recent report showed that results of commercially available ELISA assays for osteopontin in neck, head and cervix cancer patents are dependent upon the assay kit used (Vordermark, et al, “Plasma osteopontin levels in patients with head and neck cancer and cervix cancer are critically dependent on the choice of ELISA system” BMC Cancer, 6(1):207, 2006). The authors did not consider that the differences between the assays may be due to the detection (or lack of detection) of the specific osteopontin fragments that correlate with disease presence, progression and prognosis.
Therefore, what is needed are reagents and methods that will specifically detect osteopontin fragments and distinguish them from each other and from the full-length protein.